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Growth of Newcastle disease virus in fertile eggs of the Japanese quail (Coturnix coturnix japonica)
J. COMP.
PATH.
1972.
GROWTH
VOL.
OF
82.
87
NEWCASTLE
EGGS
DISEASE
OF THE
(COTURNIX
VIRUS
JAPANESE
COTURNIX
IN
FERTILE
QUAIL JAPONICA)
BY
D. A. Department
of Agriculture
HIGGINS*
and Fisheries, Castle Peak Veterinary Hong Kong
Laboratory,
New Territories,
INTRODUCTION
Initial studies on virus culture in fertile eggs of the domestic fowl (Gallus dome&us) were made in 1911 by Rous and Murphy. Today, the fertile egg technique is still the most efficient method for growth of many viruses. The literature concerning culture of Newcastle disease virus (NDV) in fertile eggs was reviewed by Lancaster (1966) and Bang (1964). Apart from some attempts to grow NDV in fertile duck eggs (Collier and Dinger, 1950; Seetharaman, 1951), only the chick embryo among the many avian species has been widely used. Recent observations (Higgins and Wong, 1968; Hashimoto, Kaneko, Onoshi, Kobayashi, Maeda and Kita, 1969) showing that the Japanese quail (Coturnix cotu7ni.x japonica) is naturally susceptible to NDV prompted the present study in which fertile quail eggs were compared with fertile hen eggs for the culture of five strains of NDV.
MATERIALS
AND
METHODS
Fertile eggs. Hen eggs were obtained from a healthy, vaccinated flock of White Leghorn fowls maintained at this station. Quail eggswere purchased from a healthy, unvaccinated, commercial laying flock. Virus strains. Two vaccine strains, F and Ranikhet, and three Hong Kong field strains, designated A, B and C, were used. Haemagglutinution (HA) test. The technique described in Methods for the Examination of Poultry Biologics (Anon., 1963) was used. Virus titration in fertile eggs. A mean embryo infective dose (EID,,) titration was performed as described in Methods for the Examination of Poultry Biologics (Anon., 1963). Eggs were candled 18, 42, 56, 70, 94 and 118 hours after incubation. Allantoic fluids of dead embryos and of embryos alive at 118 hours were slide-tested against fowl red blood cells (FRBC) and eggs containing agglutinins were considered to be infected with NDV. The fifty per cent. end-point of infectivity was calculated by the method of Reed and Muench (1938). The embryo death times after inoculation recorded during the EIDbo titration for embryos shown to be infected with NDV were used to calculate the mean embryo death time (EDT,,). *Present University,
address: Department N.Y. 14850, U.S.A.
of Microbiology,
New
York
State
Veterinary
College,
Cornell
88
N.D.
VIRUS
IN
JAPANESE
QUAIL
EGGS
Experimental method. Before quail eggs were set for incubation their shellswere decolourised by rubbing with cotton swabssoaked in oxalic acid; the acid was then neutralised by dipping the egg in ethyl alcohol. Hen eggs were inoculated on the ninth day and quail eggson the sixth day of incubation. At these times the embryos of the two specieshad achieved approximately the samedegree of development relative to the size of the egg, as judged by routine candling. Each virus strain was passagedonce through hen eggs by the allantoic sac route, and the infected allantoic fluids were collected. These seed cultures were examined by HA tests and each was titrated in quail eggs and in hen eggs; the EID,, and EDT,, were then calculated. All egg inoculations were done on the same day and one dilution seriesof each virus was used for inoculation of quail and hen eggs. All allantoic fluids subsequently shown by the slide agglutination test to contain FRBC agglutinins were examined by HA test. Allantoic fluids from dead embryos, but which did not agglutinate FRBC, were examined by bacterial culture; if the allantoic fluid was sterile the embryo death was classified as “non-specific”. Allantoic fluids of infected eggs initially inoculated with a lo3 dilution of seed virus were pooled in equal volumes, one pool for each titration. The resulting ten virus pools were then examined by EID50 titration in fertile hen eggs.
RESULTS
Table 1 summarises the results. The EIDao were higher and the EDTao were shorter in quail eggs than in hen eggs. The F strain of NDV killed all the quail embryos, but hardly any chick embryos. Non-specific deaths were generally higher among quail embryos than among chick embryos. The virus content of infected quail embryo allantoic fluids was lower than that of chick embryo allantoic fluids, as assessed by HA and EIDao tests,
DISCUSSION
The present investigation showed that quail embryos were more susceptible than chick embryos to NDV. It might have been expected that specific antibodies in the hen egg yolk could have reduced the pathogenicity of the viruses : nevertheless, during the second week of incubation, parentally acquired antibodies seem to have little effect on the growth of the NDV in the allantois (Hitchner, Reising and Van Roekel, 1951; Higgins, 1970) probably because yolk sac antibodies do not diffuse throughout the egg until the third week of incubation (Bang, 1964). If yolk sac antibodies had influenced growth of NDV in hen eggs, lower HA and EIDao titres would have been expected from chick than from quail embryo allantoic fluids. The opposite was found, however, possibly due to the shorter time for virus growth in quail eggs before embryo death (Daniel and Hanson, 1968). The smaller size of the quail egg also might affect the ultimate virus titre. Large hen eggs which yield higher HA and EIDso titres than small hen eggs
(Ablashi,
Chang and Yates, 1966) probably
contain
more cells, but at certain
stages of incubation the situation is complicated by the development of double layers of cells in the allantois, and this affects the accessibility of the cells to virus (Cairns and Fazekas de St. Groth, 1957). A direct comparison of the six-day quail embryo and the nine-day chick embryo in terms of the number of cells available for NDV replication is therefore unrealistic. Although a higher ratio
128
64
2.56
A
B
C
Hen Quail
Hen Quail
Hen Quail
Hen Quail
Hen Quail
FIVE
6.54 9.00
6.12 8.16
7.83 8.40
4.84 8.79
7.33 9-00
EID,,t
OF
___--
OF 1‘ITKATION
TABLE
1
-
708.9 128.4
344.1 80.8
701.7 158.5
1608.0 208.5
2811.5 482 .O
Mean
HA *
882048 2-256 ___28192 2-512
28192 4-2048
128-4096 8-512
4-16384 2-4096
Range
of harveJt
Results
55.6 49.3
51.6 46.0
50.6 44.8
48.0 35.0
>llO.O 65.0
EDTL, (hours)
of titration
STRAINS OF NDV IN FERTlLE HEN AND ALLANTOIC FLUIDS BY HA AND EIDB,,
as reciprocal of tine. as the reciprocal of the log, in whole numbers, of that dilution containing as the percentage of infected embryos dead by 118 hours after inoculation. as a percentage of the total eggs inoculated.
256
Ranikhet
* Expressed t Expressed $ Expressed 9: Expressed
512
F
seed
of
HA *
RESULTS
AND
;:;
6.7 8.3
13.3 16.7
15.0 15.0
A.“6.
o/O nonspeciJ;c deaths §
ASSESSMENT
the 50 per cent. infective
100 100
100 100
100 100
100 100
17.9 100
Sb embryo mortality:
QUA”, EGGS, TITRATIONS
---
of IO3
8-256 2-256
128-1024 32-64
32-1024 4-128
512-2048 8-64
4-4096 32-128
Range
dose (one EID,,).
114.0 112.5
409.6 40.0
1408.0 42.0
1946.4 138.7
Mean
HA*
Assessment
OF HAR\‘F.BI‘ED
-
9.50 9.30
9.13 8.68
7.87 780
10.17 9-00
9.37 8.38
E&ot in hen ew
harvest
90
N.D.
VIRUS
IN
JAPANESE
QUAIL
EGGS
of virus particles to susceptible cells might have effectively increased the infectivity of the inocula to quail embryos it would not necessarily explain the increased virulence of the virus strains reflected in the shorter EDTao and the lethal effect of the lentogenic F strain of NDV. It would seem, therefore, that the quail embryo is naturally more susceptible than the chick embryo to NDV. The higher number of non-specific deaths among quail embryos was probably due to the relatively greater trauma of inoculation, and might be overcome with improvement in technique. The possibility cannot be overlooked that viruses indigenous to either flock passed to the eggs and affected the non-specific death rate or altered culture patterns by interference. There was, however, no clinical evidence or history of infection in either flock, and the hatchability of over 50 uninoculated eggs from each flock was above 85 per cent. Fertile quail eggs have two main disadvantages for routine virological work : the small quantity of allantoic fluid reduces the virus yield per egg and the pigmented shells make candling difficult. In the present work the shell colour was overcome by bleaching, a procedure shown in preliminary studies to have no significant effect on percentage fertility or hatchability. In spite of these disadvantages the fertile quail egg could prove useful in the virological laboratory. The eggs take up little space and require shorter incubation times than the fertile hen egg, both prior to and during virus culture. Their susceptibility to lentogenie NDV could prove especially useful. A further advantage lies in the simplicity of the husbandry techniques applied to quails. A flock of quails adequate for laboratory purposes can be maintained in a small room, and therefore could easily be raised pathogen-free, a frequent requisite of the avian virologist. SUMMARY
Five strains of Newcastle disease virus (NDV) were inoculated into the allantoic cavities of six-day fertile quail eggs and nine-day fertile hen eggs. The mean embryo infective dose (EIDw) was higher and the mean embryo death time (EDTIo) was shorter in quail eggs than in hen eggs. The F strain killed all the quail embryos, but hardly any chick embryos. The HA and EIDM titres of allantoic fluids were higher from hen eggs than from quail eggs. It is considered that fertile quail eggs have some advantages over hen eggs for use in the virological laboratory. REFERENCES
-
(1963). Methods for the Examination of Poultry Biologics, 2nd Edit. (revised). Publication 1038, National Academy of Sciences, National Research Council; Washington, D.C. Ablashi, D. V., Chang, P. W., and Yates, V. J. (1966). Asian Diseases, 10, 224. The Bang, F. B. (1964). Newcade Disease Virus, Ed. R. P. Hanson, pp. 247-256, University of Wisconsin Press. Cairns, H. J. F., and Fazekas de St. Groth (1957). J. Immunol., 78, 191. Collier, W. A., and Dinger, J. E. (1950). Dot. need. Ind. Morb. Trap., 2, 189. Daniel, M. D., and Hanson, R. P. (1968). Asian Diseases, 12, 434. Hashimoto, K., Kaneko, S., Onoshi, H., Kobayashi, K., Maeda, M., and Kita, E. (1969). Bull. Nat. Inst. Anim. Hlth., No. 58, 7.
D.
A.
91
HIGGINS
Higgins, D. A. (1970). Asian Diseases, 14, 579. Higgins, D. A., and Wong, F. S. F. (1968). I/et. Rec., 83, 437. Hitchner, S. B., Reising, G., and Van Roekel (1951). Amer. J. vet. Res., 12, 246. Lancaster, J. E. (1966). Newcastle Disease, Monograph No. 3, Health of Animals Branch; Canada Department of Agriculture. Reed, L. V., and Muench, H. (1938). Amer. J. Hyg., 27, 493. Rous, P., and Murphy, J. B. (1911). J. Amer. med. Ass., 56, 741. Seetharaman, C. (1951). Indian vet. J., 37, 331. [Received
for
publication,
March
3rd, 19711
PATH.
1972.
GROWTH
VOL.
OF
82.
87
NEWCASTLE
EGGS
DISEASE
OF THE
(COTURNIX
VIRUS
JAPANESE
COTURNIX
IN
FERTILE
QUAIL JAPONICA)
BY
D. A. Department
of Agriculture
HIGGINS*
and Fisheries, Castle Peak Veterinary Hong Kong
Laboratory,
New Territories,
INTRODUCTION
Initial studies on virus culture in fertile eggs of the domestic fowl (Gallus dome&us) were made in 1911 by Rous and Murphy. Today, the fertile egg technique is still the most efficient method for growth of many viruses. The literature concerning culture of Newcastle disease virus (NDV) in fertile eggs was reviewed by Lancaster (1966) and Bang (1964). Apart from some attempts to grow NDV in fertile duck eggs (Collier and Dinger, 1950; Seetharaman, 1951), only the chick embryo among the many avian species has been widely used. Recent observations (Higgins and Wong, 1968; Hashimoto, Kaneko, Onoshi, Kobayashi, Maeda and Kita, 1969) showing that the Japanese quail (Coturnix cotu7ni.x japonica) is naturally susceptible to NDV prompted the present study in which fertile quail eggs were compared with fertile hen eggs for the culture of five strains of NDV.
MATERIALS
AND
METHODS
Fertile eggs. Hen eggs were obtained from a healthy, vaccinated flock of White Leghorn fowls maintained at this station. Quail eggswere purchased from a healthy, unvaccinated, commercial laying flock. Virus strains. Two vaccine strains, F and Ranikhet, and three Hong Kong field strains, designated A, B and C, were used. Haemagglutinution (HA) test. The technique described in Methods for the Examination of Poultry Biologics (Anon., 1963) was used. Virus titration in fertile eggs. A mean embryo infective dose (EID,,) titration was performed as described in Methods for the Examination of Poultry Biologics (Anon., 1963). Eggs were candled 18, 42, 56, 70, 94 and 118 hours after incubation. Allantoic fluids of dead embryos and of embryos alive at 118 hours were slide-tested against fowl red blood cells (FRBC) and eggs containing agglutinins were considered to be infected with NDV. The fifty per cent. end-point of infectivity was calculated by the method of Reed and Muench (1938). The embryo death times after inoculation recorded during the EIDbo titration for embryos shown to be infected with NDV were used to calculate the mean embryo death time (EDT,,). *Present University,
address: Department N.Y. 14850, U.S.A.
of Microbiology,
New
York
State
Veterinary
College,
Cornell
88
N.D.
VIRUS
IN
JAPANESE
QUAIL
EGGS
Experimental method. Before quail eggs were set for incubation their shellswere decolourised by rubbing with cotton swabssoaked in oxalic acid; the acid was then neutralised by dipping the egg in ethyl alcohol. Hen eggs were inoculated on the ninth day and quail eggson the sixth day of incubation. At these times the embryos of the two specieshad achieved approximately the samedegree of development relative to the size of the egg, as judged by routine candling. Each virus strain was passagedonce through hen eggs by the allantoic sac route, and the infected allantoic fluids were collected. These seed cultures were examined by HA tests and each was titrated in quail eggs and in hen eggs; the EID,, and EDT,, were then calculated. All egg inoculations were done on the same day and one dilution seriesof each virus was used for inoculation of quail and hen eggs. All allantoic fluids subsequently shown by the slide agglutination test to contain FRBC agglutinins were examined by HA test. Allantoic fluids from dead embryos, but which did not agglutinate FRBC, were examined by bacterial culture; if the allantoic fluid was sterile the embryo death was classified as “non-specific”. Allantoic fluids of infected eggs initially inoculated with a lo3 dilution of seed virus were pooled in equal volumes, one pool for each titration. The resulting ten virus pools were then examined by EID50 titration in fertile hen eggs.
RESULTS
Table 1 summarises the results. The EIDao were higher and the EDTao were shorter in quail eggs than in hen eggs. The F strain of NDV killed all the quail embryos, but hardly any chick embryos. Non-specific deaths were generally higher among quail embryos than among chick embryos. The virus content of infected quail embryo allantoic fluids was lower than that of chick embryo allantoic fluids, as assessed by HA and EIDao tests,
DISCUSSION
The present investigation showed that quail embryos were more susceptible than chick embryos to NDV. It might have been expected that specific antibodies in the hen egg yolk could have reduced the pathogenicity of the viruses : nevertheless, during the second week of incubation, parentally acquired antibodies seem to have little effect on the growth of the NDV in the allantois (Hitchner, Reising and Van Roekel, 1951; Higgins, 1970) probably because yolk sac antibodies do not diffuse throughout the egg until the third week of incubation (Bang, 1964). If yolk sac antibodies had influenced growth of NDV in hen eggs, lower HA and EIDao titres would have been expected from chick than from quail embryo allantoic fluids. The opposite was found, however, possibly due to the shorter time for virus growth in quail eggs before embryo death (Daniel and Hanson, 1968). The smaller size of the quail egg also might affect the ultimate virus titre. Large hen eggs which yield higher HA and EIDso titres than small hen eggs
(Ablashi,
Chang and Yates, 1966) probably
contain
more cells, but at certain
stages of incubation the situation is complicated by the development of double layers of cells in the allantois, and this affects the accessibility of the cells to virus (Cairns and Fazekas de St. Groth, 1957). A direct comparison of the six-day quail embryo and the nine-day chick embryo in terms of the number of cells available for NDV replication is therefore unrealistic. Although a higher ratio
128
64
2.56
A
B
C
Hen Quail
Hen Quail
Hen Quail
Hen Quail
Hen Quail
FIVE
6.54 9.00
6.12 8.16
7.83 8.40
4.84 8.79
7.33 9-00
EID,,t
OF
___--
OF 1‘ITKATION
TABLE
1
-
708.9 128.4
344.1 80.8
701.7 158.5
1608.0 208.5
2811.5 482 .O
Mean
HA *
882048 2-256 ___28192 2-512
28192 4-2048
128-4096 8-512
4-16384 2-4096
Range
of harveJt
Results
55.6 49.3
51.6 46.0
50.6 44.8
48.0 35.0
>llO.O 65.0
EDTL, (hours)
of titration
STRAINS OF NDV IN FERTlLE HEN AND ALLANTOIC FLUIDS BY HA AND EIDB,,
as reciprocal of tine. as the reciprocal of the log, in whole numbers, of that dilution containing as the percentage of infected embryos dead by 118 hours after inoculation. as a percentage of the total eggs inoculated.
256
Ranikhet
* Expressed t Expressed $ Expressed 9: Expressed
512
F
seed
of
HA *
RESULTS
AND
;:;
6.7 8.3
13.3 16.7
15.0 15.0
A.“6.
o/O nonspeciJ;c deaths §
ASSESSMENT
the 50 per cent. infective
100 100
100 100
100 100
100 100
17.9 100
Sb embryo mortality:
QUA”, EGGS, TITRATIONS
---
of IO3
8-256 2-256
128-1024 32-64
32-1024 4-128
512-2048 8-64
4-4096 32-128
Range
dose (one EID,,).
114.0 112.5
409.6 40.0
1408.0 42.0
1946.4 138.7
Mean
HA*
Assessment
OF HAR\‘F.BI‘ED
-
9.50 9.30
9.13 8.68
7.87 780
10.17 9-00
9.37 8.38
E&ot in hen ew
harvest
90
N.D.
VIRUS
IN
JAPANESE
QUAIL
EGGS
of virus particles to susceptible cells might have effectively increased the infectivity of the inocula to quail embryos it would not necessarily explain the increased virulence of the virus strains reflected in the shorter EDTao and the lethal effect of the lentogenic F strain of NDV. It would seem, therefore, that the quail embryo is naturally more susceptible than the chick embryo to NDV. The higher number of non-specific deaths among quail embryos was probably due to the relatively greater trauma of inoculation, and might be overcome with improvement in technique. The possibility cannot be overlooked that viruses indigenous to either flock passed to the eggs and affected the non-specific death rate or altered culture patterns by interference. There was, however, no clinical evidence or history of infection in either flock, and the hatchability of over 50 uninoculated eggs from each flock was above 85 per cent. Fertile quail eggs have two main disadvantages for routine virological work : the small quantity of allantoic fluid reduces the virus yield per egg and the pigmented shells make candling difficult. In the present work the shell colour was overcome by bleaching, a procedure shown in preliminary studies to have no significant effect on percentage fertility or hatchability. In spite of these disadvantages the fertile quail egg could prove useful in the virological laboratory. The eggs take up little space and require shorter incubation times than the fertile hen egg, both prior to and during virus culture. Their susceptibility to lentogenie NDV could prove especially useful. A further advantage lies in the simplicity of the husbandry techniques applied to quails. A flock of quails adequate for laboratory purposes can be maintained in a small room, and therefore could easily be raised pathogen-free, a frequent requisite of the avian virologist. SUMMARY
Five strains of Newcastle disease virus (NDV) were inoculated into the allantoic cavities of six-day fertile quail eggs and nine-day fertile hen eggs. The mean embryo infective dose (EIDw) was higher and the mean embryo death time (EDTIo) was shorter in quail eggs than in hen eggs. The F strain killed all the quail embryos, but hardly any chick embryos. The HA and EIDM titres of allantoic fluids were higher from hen eggs than from quail eggs. It is considered that fertile quail eggs have some advantages over hen eggs for use in the virological laboratory. REFERENCES
-
(1963). Methods for the Examination of Poultry Biologics, 2nd Edit. (revised). Publication 1038, National Academy of Sciences, National Research Council; Washington, D.C. Ablashi, D. V., Chang, P. W., and Yates, V. J. (1966). Asian Diseases, 10, 224. The Bang, F. B. (1964). Newcade Disease Virus, Ed. R. P. Hanson, pp. 247-256, University of Wisconsin Press. Cairns, H. J. F., and Fazekas de St. Groth (1957). J. Immunol., 78, 191. Collier, W. A., and Dinger, J. E. (1950). Dot. need. Ind. Morb. Trap., 2, 189. Daniel, M. D., and Hanson, R. P. (1968). Asian Diseases, 12, 434. Hashimoto, K., Kaneko, S., Onoshi, H., Kobayashi, K., Maeda, M., and Kita, E. (1969). Bull. Nat. Inst. Anim. Hlth., No. 58, 7.
D.
A.
91
HIGGINS
Higgins, D. A. (1970). Asian Diseases, 14, 579. Higgins, D. A., and Wong, F. S. F. (1968). I/et. Rec., 83, 437. Hitchner, S. B., Reising, G., and Van Roekel (1951). Amer. J. vet. Res., 12, 246. Lancaster, J. E. (1966). Newcastle Disease, Monograph No. 3, Health of Animals Branch; Canada Department of Agriculture. Reed, L. V., and Muench, H. (1938). Amer. J. Hyg., 27, 493. Rous, P., and Murphy, J. B. (1911). J. Amer. med. Ass., 56, 741. Seetharaman, C. (1951). Indian vet. J., 37, 331. [Received
for
publication,
March
3rd, 19711